The present invention refers to the use of a trichothecene-transforming alcohol dehydrogenase, a procedure for the transformation of trichothecenes, and a trichothecene-transforming additive.
Trichothecenes represent a frequently occurring group of mycotoxins that includes deoxynivalenol (DON, CAS no. 51481-10-8), T-2 toxin (CAS no. 21259-20-1), HT-2 toxin (CAS no. 26934-87-2), nivalenol (CAS no. 23282-20-4), fuseranon-X (CAS no. 23255-69-8), scripentriol, 15-acetoxyscirpenol (CAS no. 2623-22-5), 4,15-diacetoxyscirpenol (CAS no. 2270-40-8), trichodermol (CAS no. 2198-93-8), verrucarin A (CAS no. 3148-09-2), verrucarin J (CAS no. 4643-58-7), isotrichodermin (CAS no. 91423-90-4), hydroxyisotrichodermin (CAS no. 344781-02-8), calonectrin (CAS no. 38818-51-8), T-2 tetraol (CAS no. 34114-99-3), deacetylneosolaniol (CAS no. 74833-39-9), neosolaniol (CAS no. 36519-25-2), acetylneosolaniol (CAS no. 65041-92-1), sporotrichiol (CAS no. 101401-89-2), trichotriol (CAS no. 109890-37-1), sambucinol (CAS no. 90044-33-0), and culmorin (CAS no. 18374-83-9), among others. Trichothecenes, particularly DON, also known as vomitoxin, can be produced by a number of Fusarium fungi, especially F. graminearum and F. culmorum. These fungi attack crops such as maize, various types of grain, such as wheat, oats, or barley, whereas usually the fungal attack occurs before harvest and the fungal growth or mycotoxin formation can also occur before, or in the case of improper storage, after harvest.
The Food and Agriculture Organization (FAO) estimates that worldwide 25% of agricultural products are contaminated with mycotoxins, which results in considerable economic losses. In a more current study carried out worldwide by I. Rodrigues and K. Naehrer, Toxins, 2012, 4, 663-675, during a time period from January 2009 to December 2011, a total of 23,781 samples were analysed, of which 81% tested positive for at least one mycotoxin and 59% tested positive for trichothecenes, especially DON. Trichothecenes, especially DON could be found with a frequency of up to 100% in all regions of the world, as well as in all grain and feed classes tested, such as maize, soya meal, wheat, wheat bran, DDGS (distiller's dried grains with solubles), and in prepared feed mixtures. Apart from basic, non-processed foodstuffs, evidence of trichothecenes was also found in processed foods, such as flour, breakfast cereal, pasta products, bread, pastry, and wheat-based children's and baby food.
Trichothecenes have the following structural formula:
wherein the different substitution remainders R1 to R5 differ depending on the type of trichothecene. It is a known fact that, in addition to the epoxy group, an intact alpha-hydroxy group on the C-3 atom of the trichothecenes is jointly responsible for their toxic effect. Trichothecene types with a hydroxy group on the C-3 atom include deoxynivalenol, T-2 toxin, HT-2 toxin, nivalenol, fuseranon-X, 15-acetoxyscirpenol, 4,15-diacetoxyscirpenol, trichodermol, T-2 tetraol, deacetylneosolaniol, acetylneosolaniol, sporotrichiol, trichotriol, sambucinol, and culmorin.
Deoxynivalenol (DON) has a characteristic carbonyl group on the C-8 atom and has the following structural formula:
and the IUPAC name (3α,7α)-3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one. In nature, several toxic DON subtypes also occur with a hydroxy group on the C-3 atom. Examples of these are acetylated DON (e.g. 15 acyl DON), glycosylated DON, DON sulfonate (e.g. DONS-1, DONS-2), or DON sulfate (DON 15 sulfate). These DON subtypes also belong to the trichothecene types with a hydroxy group or substituted hydroxy group on the C-3 atom.
Because of the toxic effect of DON, limits or maximum levels have been defined by the competent authorities for food and feed. Thus the European Union has regulated the DON content in food (EC no. 1881/2006, EC no. 1126/2007) and has recommended maximum levels for feed (2006/576/EC). In the USA, the FDA has published maximum levels.
Illnesses that are caused by ingesting mycotoxins in humans or animals are referred to as mycotoxicoses. In the case of trichothecenes or trichothecene types, these are also referred to as “trichothecene mycotoxicoses”, more specifically as “mycotoxicoses caused by trichothecenes exhibiting a hydroxyl group on the C-3 atom”, or even more specifically as “DON mycotoxicoses”. It is a known fact that the toxic effects of trichothecenes on animals and humans are based on several factors. These factors include the inhibition of protein biosynthesis, possible interaction with serotonin and dopamine receptors, and the upregulation of proinflammatory cytokines (EFSA Journal 2004, 73, 1-41). Moreover, DON mycotoxicoses cause changes in biomarkers, as diagnosed by an increase in the IgA concentration in blood, an increase in the SOCS3 concentration in the liver, or the reduction of IGFALS levels in the plasma (Pestka et al. 2004, Toxicol. Lett. 153, 61-73) as well as a reduction of the claudin concentration in the intestines (Pinton et al. 2009, Tox. Appl. Pharmacol. 237, 41-48).
For example, trichothecene mycotoxicoses are exhibited in swine by reduced feed intake, reduced growth, the occurrence of vomiting and diarrhea, as well as an immunological dysfunction and impaired nutrient absorption in the intestines. In the case of poultry, trichothecene mycotoxicoses cause a deterioration in feed intake, less weight gain, incidences of diarrhea, and a reduction in the weight of eggshells, among other things. In the case of ruminants, reduced feed intake and less milk production were described. In aquaculture, trichothecene mycotoxicoses cause a deterioration of feed intake and of growth rates in fish (e.g. salmon, catfish, or trout) and shrimp, among other things (Binder et. al, Guide to Mykotoxins; ISBN 978-0-9573721-0-8). Toxic effects have also been described in dogs and cats (EFSA Journal 2004, 73, 1-41). In humans, trichothecene mycotoxicoses can cause nausea, vomiting, diarrhea, abdominal pains, headache, or fever, among other things (Sobrova et. al, Interdisc. Toxicol. 2010, 3 (3), 94-99).
The primary strategy for the reduction of a trichothecene or DON contamination of food or feed is the restriction of fungal attack, for example, by complying with “good agricultural practice”. This includes the use of seeds that are free of parasites and fungus, or the ploughing-in of crop residues. Moreover, fungal growth in the field can be reduced by the correct use of fungicides. After harvest, the crops should be stored at a residual humidity below 15% and at a low temperature to prevent fungal growth. Likewise, crops contaminated by fungal infestation should be removed before any further processing. Despite this list of measures, I. Rodriges and K. Naehrer reported (in 2012) that even in regions with the highest agricultural standards like the USA and Central Europe, 79% or 72% of all maize samples tested from 2009 to 2011 were contaminated with DON.
Other options for reducing mycotoxin contamination in food or feed are their adsorption or transformation. For adsorption, it is necessary for the binding of the mycotoxin to the adsorbent to be strong and specific over a wide pH range and that it remains stable in the gastrointestinal area during the entire digestion process. Although some non-biological adsorbents like activated carbon, silicates, or synthetic polymers like cholestyramine can be used efficiently for aflatoxins, their use for other mycotoxins, especially for trichothecenes, is not effective. Biological adsorbents such as yeast or yeast extracts are also described in the literature, but have a limitation similar to that of non-biological adsorbents. A substantial disadvantage of adsorbents is their possible non-specific bonding of other molecules that can be essential for nutrition.
Also the transformation, especially the detoxification of trichothecenes by physical and chemical treatments is limited because DON is very stable and remains stable even at heat treatments of up to 350° C.
A possible microbial transformation of DON was described in the EP-B 1 042 449, according to which the microorganism BBSH 797 (DSM 11798) is used for the detoxification of DON. Here the detoxification is based on the opening of the epoxide ring on the C-12 and C-13 atoms of DON. US 2012/0263827 A describes the biotransformation of DON to 3-epi-DON by a microorganism with the international Canadian accession number 040408-1. For many technical feed or food processes, however, an admixture of microorganisms or adsorbents is not possible, or is not legally permitted, so that there a transformation or a detoxification of trichothecenes like DON or DON subtypes is not possible.
Trichothecenes like DON and DON subtypes are absorbed rapidly into the gastrointestinal tract of human or animal bodies, which is why a fast and targeted detoxification is important.
The alcohol dehydrogenase of SEQ ID no. 1 was first described in the JP-A 2003/159079 for the production of 2-ketogulonic acid. WO 2009/133464 describes a process for the oxidation of saccharides by means of the enzyme of SEQ ID no. 1 in food and feed for the oxidation of starch, especially in the baking industry, to slow down the ageing processes in bread. Here, alcohol dehydrogenase is used for the oxidation of hydroxyl groups of carbohydrates.
Alcohol dehydrogenases with SEQ ID numbers 2 and 3 were identified in the course of a genome sequencing of Devosia sp. microorganisms and are stored online in the server of the National Center for Biotechnology Information (NCBI) under identification numbers GI:737041022 and GI:630002266. A more accurate characterisation of the alcohol dehydrogenases with SEQ ID numbers 2 and 3 was not given in the course of this work.
Because of the variety of toxic effects of trichothecenes and the frequency of their occurrence, there is therefore a need for substances or groups of substances like enzymes that can be used for the specific, safe, and permissible transformation, especially detoxification of trichothecenes.